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Immunoreactive Localization of Transforming Growth Factor-β Type II Receptor-Positive Cells in Rat Tibiae
Bone Vol. 22, No. 2 February 1998:93–98
ORIGINAL ARTICLES
Immunoreactive Localization of Transforming Growth Factor-b Type II Receptor-Positive Cells in Rat Tibiae Y. KABASAWA,1,2 S. EJIRI,2 Y. MATSUKI,1 K. HARA,1 and H. OZAWA2 Departments of 1Periodontology and 2Oral Anatomy I, Niigata University, School of Dentistry, Niigata, Japan
TGF-b type II receptor is a transmembrane serine/threonine kinase that, upon ligand binding, recruits and phosphorylates a second transmembrane kinase, the type I receptor in the GS domain, as a requirement for signal transduction.14,29 Phosphorylation allows the receptor to propagate the signal to downstream substrates.14,29,30 In the organogenesis of the mouse embryo, TGF-b type II receptor mRNA expression was detected in the undifferentiated supporting mesenchyme in the developing limb and in the vertebral column.13,21 By immunohistochemical observation during mouse organogenesis, the TGF-b type II receptor localized in Meckel’s chondrocytes, in undifferentiated mesenchymal cells around Meckel’s cartilage, and the cartilage and in the mesenchyme in the base of the skull, which subsequently differentiated into bone.8 In chimeric mice using a TGF-b type II receptor (2/2) embryonic stem cell line, TGF-b type II receptor has been reported to be important for normal development in a variety of organs, due to the several types of congenital abnormalities it reveals.20 We investigated the ligand-binding receptor, the TGF-b type II receptor in postnatal rat tibiae, by immunohistochemistry, RT-PCR, and in situ hybridization, and examined, in particular, the role of TGF-b in normal bone tissue.
To identify the target cells of transforming growth factor-b (TGF-b) in normal bone tissue, we examined TGF-b type II receptor expression using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization in young rat tibiae. In the epiphyseal growth plate, the TGF-b type II receptor cDNA was detected by RT-PCR and, alternatively, the TGF-b type II receptor protein and mRNA expression were observed in the chondrocytes in the lower part of the proliferative cell layer, and in maturative and hypertrophic cell layers by immunohistochemistry and in situ hybridization. Of these, proliferative and maturative chondrocytes, in particular, revealed strong mRNA expression. In the cortical bone area, immunoreactivity for the TGF-b type II receptor was detected in the fibroblastic cells near the osteoblasts on the endosteal surface of cortical bone. In conclusion, our findings suggest that target cells of TGF-b in normal bone tissue could be considered mainly as extracellular matrix-producing chondrocytes and undifferentiated preosteoblasts, and TGF-b may affect matrix production and differentiation of these cells. (Bone 22:93–98; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: TGF-b type II receptor; Immunohistochemistry; In situ hybridization; RT-PCR; Rat tibiae; Growth plate.
Materials and Methods
Introduction
3-week-old male Wistar rats, weighing approximately 56.8 g (n 5 11), were anesthetized with 40 mg/kg body weight sodium pentobarbital, and perfused with 4% paraformaldehyde in a 0.1 mol/L phosphate buffer (pH 7.4) for 15 min. Tibiae were dissected and immersed in the aforementioned fixative at 4°C for 2– 8 h, then decalcified with 10% EDTA (pH 7.3) at 4°C for 1 week. Specimens were dehydrated with an increasing concentration of ethanol and embedded in paraffin. Serial sagittal sections were obtained at a thickness of 6 mm and mounted on poly-Llysine-coated slides. Sections were used for hematoxylin-eosin staining.
Tissue Preparation
TGF-b is one of the most abundant growth factors in bone, and may play a critical role in bone remodeling and cartilage growth and metabolism.2,4 An in vivo TGF-b1 injection to the parietal bone of neonatal rats stimulated intramembranous ossification,17,25 whereas an injection to newborn rat femurs promoted endochondral ossification.11 In experimental defects of mature rat calvariae, grafting of TGF-b-coated b-tricalcium-phosphate pellets promoted new bone formation.19 Joyce et al. suggested that TGF-b was synthesized by osteoblasts and chondrocytes throughout rat bone fracture healing.10 In vitro immunohistochemical study of mouse bone tissue showed that immunoreactivity for TGF-b was present in the sealing zone covered by osteoclasts in the bone resorption lacunae and in the extracellular matrix adjacent to the osteoclasts.7
Immunohistochemical Study Immunohistochemistry was carried out according to Hoshi et al.6 with some modifications. To inhibit endogenous peroxidase, the deparaffinized sections were treated with 100% methanol containing 0.3% hydrogen peroxide for 1 h. The sections were immersed in phosphate-buffered saline (PBS) containing 10% low-fat milk protein for 1 h to block nonspecific binding in the tissue, and then incubated with IgG fractions of anti-TGF-b type II receptor (UBI, Lake Placid, NY) at a concentration of approx-
Address for correspondence and reprints: Kohji Hara, D.D.S., Ph.D., Department of Periodontology, Niigata University, School of Dentistry, Gakkocho 2-5274, Niigata 951, Japan. E-mail: [email protected]. ac.jp © 1998 by Elsevier Science Inc. All rights reserved.
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imately 6 mg/mL overnight. Following washing with PBS, sections were incubated with horseradish peroxidase-conjugated goat, antirabbit IgG (Dako, Glostrup, Denmark) diluted to 1:100 at room temperature for 2 h. Immunoreaction was visualized by incubation in 0.05 mol/L Tris-HCl buffer (pH 7.6) containing 0.05% 39,3-diaminobenzidine (Dojin Co., Kumamoto, Japan), 0.066% sodium azide (Wako Chemicals, Osaka, Japan), and 0.01% hydrogen peroxide. The sections were counterstained with hematoxylin. Negative control specimens were reacted with normal rabbit IgG (Dako) at 6 mg/mL instead of primary antibody. RNA Isolation, RT-PCR Total cellular RNA was extracted from the epiphyseal growth plate of the tibiae of 3-week-old Wistar rats (n 5 20). Epiphyseal growth plates were excised from the tibiae using a dissecting microscope. Total RNA extraction was prepared according to the manufacturer’s protocol (Isogen; Nippon gene, Toyama City, Japan).22 100 mg of tissue was mixed with 1 mL of guanidine isothiocyanate-phenol solution (Isogen), followed by the addition of 200 mL of chloroform, and then centrifuged at 12,000g at 4°C for 15 min. Subsequently, the aqueous phase containing RNA was added with 500 mL of isopropanol, kept at 220°C for 30 min, and centrifuged at 12,000g at 4°C for 10 min. The RNA pellet was diluted in 1 mL of 75% ethanol and centrifuged at 7500g for 5 min at 4°C. The recovered RNA was then resuspended in DEPC-treated H2O (DEPC: 0.1% diethylpyrocarbonate; Aldrich, Milwaukee, WI). First-strand cDNA was synthesized according to the method of Yamazaki et al.,31 using M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) and 50 mg/mL random hexanucleotides (Promega, Madison, WI) from 1 mg of total RNA in the reaction buffer (Gibco) containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, and 3 mmol/L MgCl2, supplemented with 40 U of RNase inhibitor (Toyobo, Osaka, Japan), 10 mmol/L dithiothreitol (Gibco), and dNTPs (dATP, dCTP, dGTP, and dTTP, each at 0.5 mmol/L; Takara shuzo, Shiga, Japan). The reaction mixture was incubated at 42°C for 3 h, then heated at 94°C for 5 min. Nucleotide sequences 301–322 (59-GGAAGTCTGCGTGGCCGTGTGG-39) and 578 –599 (59-CTATGGCAATCCCCAGCGGAGG-39) of the rat TGF-b type II receptor cDNA were used as PCR primers.21,27 A polymerase chain reaction was performed according to Sugita et al.22 A 2 mL volume of reverse transcription (RT) reaction mixture was added to 98 mL of PCR buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, dNTPs (each at 0.1 mmol/L; Takara shuzo), 25 pmol of each primer, and 5 U of recombinant Taq DNA polymerase (Takara shuzo). The reaction mixture was overlaid with mineral oil and subjected to PCR incubation for 38 cycles using an automated DNA thermal cycler (MJ Research, Watertown, MA). The PCR reaction profile was as follows21: 1 min denaturation at 96°C; 1 min annealing at 52°C; and 2 min primer extension at 72°C. The PCR end product was loaded on 3% NuSieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME) in 0.5 3 TBE (45 mmol/L Tris-borate, 1 mmol/L EDTA), stained with 0.5 mg/mL ethidium bromide. The DNA molecular weight marker (fX174/HinfI digest, Nippon gene) was run simultaneously. The gel was photographed with Tri-X pan 400 film for black-and-white prints (Eastman Kodak, Rochester, NY). Probes Used for In Situ Hybridization Total RNA was extracted as previously mentioned from 21 day fetal craniofacial tissue (n 5 2). RT-PCR was performed
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according to the method of Sugita et al.22 0.1 mg of total RNA was added to a reaction mixture containing 50 mmol/L TrisHCl (pH 8.3), 100 mmol/L KCl, 10 mmol/L MgCl2, 4 mmol/L dithiothreitol (Life Sciences, St. Petersburg, FL), 60 U of RNase inhibitor (Toyobo), 0.85 mmol/L dNTPs (Takara shuzo), 50 mg/mL random hexadeoxynucleotide primers (Promega), and 34.8 U of avian myeloblastosis virus reverse transcriptase (Life Sciences) in a final volume of 50 mL. After incubation at 42°C for 3 h, the mixture was heated at 94°C for 5 min, then stored at 4°C. A 2 mL volume of RT reaction mixture was added to 98 mL of PCR buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, dNTPs (each at 0.1 mmol/L; Takara shuzo), 25 pmol of each primer, and 5 U of recombinant Taq DNA polymerase (Takara shuzo). The PCR reaction profile was as follows: 1 min denaturation at 96°C; 1 min annealing at 52°C; and 2 min primer extension at 72°C, for 38 cycles. The PCR end product was loaded on 3% NuSieve 3:1 agarose gel (FMC Bioproducts) in 0.5 3 TBE at 100 V for 1.5 h, stained with 0.5 mg/mL ethidium bromide, and a molecular weight species corresponding to the target 299 bp end product was observed (data not shown). The PCR end product was subjected to electrophoresis on 3% agarose gel and then gel purified using the Qiaex Gel Extraction Kit (Qiagen, Hilden, Germany). For the direct insertion of the RT-PCR product into the plasmid vector pCR™ II, the TA Cloning Kit (Invitrogen, San Diego, CA) was used.12,22 The subcloned fragment was confirmed to be identical to published rat TGF-b type II receptor cDNA by DNA sequence analysis using an automated DNA sequencer (Model 373A, Applied Biosystems, Foster City, CA). In vitro transcription and digoxigenin UTP-labeled sense and antisense probes were prepared as described by the manufacturer (Boehringer Mannheim, Biochemica, Mannheim, Germany). In Situ Hybridization In situ hybridization was performed as described previously.15 Deparaffinized sections were washed in PBS and treated with 1.0 mg/mL proteinase K in 20 mmol/L Tris-HCl (pH 8.0) and 10 mmol/L EDTA at 37°C for 10 min. To remove endogenous alkaline phosphatase, sections were immersed in 0.2 mol/L HCl for 10 min, washed in PBS, and soaked in 0.1 mol/L triethanolamine solution (pH 8.0) containing 0.25% acetic anhydride for 10 min to inhibit nonspecific background binding. The specimens were washed with PBS, dehydrated in graded ethanol, and air dried. The probe was used for hybridization at a final concentration of 1.0 mg/mL in a hybridization mixture (50% formamide, 10 mmol/L Tris-HCl, pH 7.6, 200 mg/mL tRNA, 1 3 Denhardt’s solution, 10% dextran sulfate, 600 mmol/L NaCl, 0.25% SDS, and 1 mmol/L EDTA). The specimens were incubated with the probes at 50°C for 16 h in a 50% formamide humid chamber, then successively washed with 2 3 SSC (standard saline-citrate buffer) containing 50% formamide at 55°C for 30 min, TNE (10 mmol/L Tris-HCl, pH 8.0, 500 mmol/L NaCl, 1 mmol/L EDTA) at 37°C for 10 min, with 20 mg/mL RNase A in TNE at 37°C for 30 min, TNE at 37°C for 10 min, once with 2 3 SSC at 50°C for 20 min, and finally twice with 0.2 3 SSC at 50°C. Specific transcripts were detected with anti-digoxigenin-conjugated alkaline phosphatase antibody (Boehringer Mannheim) by the manufacturer’s protocol. The color was developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. The sections were counterstained with methylgreen.
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Figure 1. Histology of 3-week-old rat tibia (hematoxylin-eosin staining). P: proliferative chondrocyte layer; M: maturative chondrocyte layer; H: hypertrophic chondrocyte layer. Original magnification 3225.
Results Epiphyseal Growth Plate Immunohistochemistry. In the epiphyseal growth plate of 3week-old rat tibiae, four layers of chondrocytes were distinguished: resting chondrocytes; proliferative chondrocytes; maturative chondrocytes; and hypertrophic chondrocytes. Beneath the layer of hypertrophic chondrocytes, active endochondral ossification was observed (Figure 1). In the epiphyseal growth plate, immunoreactivity for the TGF-b type II receptor was detected in chondrocytes in the lower part of the proliferative cell layer, and also in maturative and hypertrophic cell layers (Figure 2A). At a higher power, immunoreactivity was seen to correspond to the cell membranes of these chondrocytes (Figure 2B). No specific immunoreactivity was detected on the control sections incubated with normal rabbit IgG (Figure 2A). RT-PCR. Using RT-PCR, TGF-b type II receptor mRNA was present in the epiphyseal growth plate of 3-week-old rat tibiae (Figure 3). By agarose-gel analysis of TGF-b type II receptor cDNA, the predicted band of 299 bp was observed. Negative control was obtained by PCR without the prior RT step, confirming that our RT-PCR yielded products derived from mRNAs. In situ hybridization. In the epiphyseal growth plate of 3week-old rat tibiae, TGF-b type II receptor mRNA expression
Figure 2. (A) Immunopositive cells for TGF-b type II receptor in epiphyseal growth plate. P: proliferative chondrocyte layer; M: maturative chondrocyte layer; H: hypertrophic chondrocyte layer. Left: photomicrograph of section using normal rabbit IgG instead of primary antibody. No specific immunoreactivity was observed. Original magnification 3360. (B) Higher power view of right side of (A), the region of the maturative chondrocyte layer. Immunoreactivity corresponding to the cell membrane of chondrocytes was detected. Arrow heads: TGF-b type II receptor-positive cells. Original magnification 3720.
was detected in chondrocytes in the lower part of the proliferative chondrocyte layer, and in the maturative and hypertrophic chondrocyte layers (Figure 4A). Of these, proliferative and maturative chondrocytes, in particular, revealed strong mRNA
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Figure 3. Agarose-gel analysis of TGF-b type II receptor cDNA. M: marker (fX174/HinfI digest, Nippon gene); S: PCR product.
expression. At a higher power, the transcription signal was observed in cytoplasm (Figure 4B). The result generally coincided with immunolocalization of the TGF-b type II receptor in the growth plate. Trabecular Bone Area and Endosteal Surface Immunohistochemistry. In the area of primary trabecular bone subjacent to the growth plate, no specific immunoreactivity was observed (Figure 5A). In the area of the endosteum, immunoreactivity for the TGF-b type II receptor was detected on fibroblastic cells adjacent to the single layer of osteoblasts on the endosteal bone surface (Figure 5B). Control slides using normal rabbit IgG showed no immunoreactivity (Figure 5A,B). As the endosteal surface, immunolocalization was also observed in fibroblastic cells near the osteoblasts or blood vessels, but not in the osteoblasts of the cortical bone area. In the periosteum, only weak staining for TGF-b type II receptor was detected in the flat cells apart from the bone surface (data not shown). In situ hybridization. TGF-b type II receptor mRNA expression was undetected in both primary trabecular bone and cortical bone. Discussion By immunohistochemistry and in situ hybridization, localization of the TGF-b type II receptor protein and mRNA-expressing cells was evident in the lower part of the proliferative chondrocyte layer, and in the maturative and hypertrophic chondrocyte layers. The proliferative and maturative chondrocytes revealed strong mRNA expression, whereas immunoreactivity for the TGF-b type II receptor was more intense in maturative and hypertrophic chondrocytes. This indicates some possible difference in time kinetics between the cytoplasmic TGF-b type II receptor mRNA expression and immunoreactivity on the cell surface during chondrocyte differentiation. In addition, the functional receptor level may be determined by posttranscriptional regulation, or a receptor recycle mechanism. Immunolocalization of TGF-b1 was observed in the proliferative and upper hypertrophic chondrocytes, and in extracellular matrices around hypertrophic chondrocytes in the growth plate of 1-month-old Long–Evans rats, suggesting that TGF-b induced chondrocyte proliferation and regulated protein synthesis.9 In the growth plate of tibiotarsi of 3-week-old chicks, TGF-b localization preceded a marked increase in type II collagen mRNA
Figure 4. (A) TGF-b type II receptor mRNA expression of epiphyseal growth plate. P: proliferative chondrocyte layer; M: maturative chondrocyte layer; H: hypertrophic chondrocyte layer. Left: photomicrograph of section using sense probe. Original magnification 3360. (B) Higher power view of right side of (A), the region of the maturative chondrocyte layer. Original magnification 3720.
expression in maturative chondrocytes, suggesting a role for TGF-b in synthesis induction of the extracellular matrix.26 In the in vitro model of rat epiphyseal chondrocyte differentiation, TGF-b1 stabilized the phenotype of the prehypertrophic epiphyseal chondrocyte by maintenance of expression of type II collagen and aggrecan core protein, and with coordinate inhibi-
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Figure 5. (A) Localization of the TGF-b type II receptor-positive cells in primary trabecular bone (TB). H: lower part of hypertrophic chondrocyte layer. Left: photomicrograph of section using normal rabbit IgG instead of primary antibody. Original magnification 31000. (B) Localization of the TGF-b type II receptor-positive cells in endosteal bone surface. Arrow heads show fibroblastic cells near the osteoblasts. OB: osteoblasts. Left: photomicrograph of section using normal rabbit IgG instead of primary antibody. Original magnification 31000.
tion of matrix metalloproteinases.1 Taken together with our results, TGF-b produced by chondrocytes and stored at the cartilage matrix would regulate the functional differentiation and matrix production of a portion of the proliferative chondrocytes, and of maturative and hypertrophic chondrocytes, via these TGF-b type II receptors in an autocrine/paracrine fashion during endochondral ossification in the growth plate. In contrast to the growth plate, in the cartilage tissue of the mouse embryo, TGF-b type II receptor mRNA and TGF-b type II receptor-positive cells were detected in the undifferentiated mesenchyme but not in the condensing chondrocytes, suggesting that TGF-b signaling through the type II receptor may play a role in the physiology of these undifferentiated cells.8,13,21 However, based on our findings, the role of TGF-b during the cartilage formation stage may be considered to differ from chondrocyte differentiation in the growth plate. Otherwise, the effect of TGF-b would occur via a different type II receptor isoform, which would be transcribed from the originally identical TGF-b type II receptor gene,5,23 because our primers could not detect these isoforms. Several in vivo and in vitro studies have shown the importance of TGF-b in bone remodeling.2,7,16,18,26,28 Immunoreactivity for TGF-b has been observed in osteoblasts and bone matrix.7,26 Concerning bone matrix protein, TGF-b has been
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found to regulate the synthesis of osteopontin, osteonectin, and collagen in cultured osteoblastic cells,18 whereas it has also been shown to inhibit differentiation-associated phenotypes such as alkaline phosphatase activity and the production of osteocalcin.2,16,18,28 Our immunohistochemical study demonstrates that TGF-b type II receptor was detected in fibroblastic cells adjacent to the osteoblasts on the endosteal bone surface. These immunopositive cells were localized in a well-defined pattern, with the fibroblastic cells particularly close to plump osteoblasts, which appeared to be actively producing bone matrix. On the other hand, no apparent staining was observed in the osteoblasts after terminal differentiation. It was reported that, in cultured MC3T3-E1 cells, TGF-b type II receptor was downregulated in differentiation, suggesting that downregulation is one of the mechanisms by which cells escape from the control of TGF-b.24 Together with our results, these data support a view that, in normal bone tissue, TGF-b would preferentially affect undifferentiated preosteoblastic cells rather than differentiated osteoblasts in regard to extracellular matrix production and differentiation. Most TGF-b response via TGF-b type II receptors may be involved in the regulation of preosteoblast function, and upregulation of the TGF-b type II receptor would be related to rapid bone remodeling. Compared to immunopositive fibroblastic cells for TGF-b type II receptors on the endosteal bone surface, fibroblastic cells adjacent to the osteoblasts did not show similar immunoreactivity in trabecular bone. Consistent with these results, Erlebacher et al. reported that, in the mouse model of osteoblast-specific overexpression of TGF-b2 from the osteocalcin promotor, longitudinal sections through the diaphysis revealed large unmineralized areas of cortical bone in transgenic bone whereas wildtype bone was entirely mineralized. There was, however, no difference in the metaphyseal trabeculation adjacent to the growth plate.3 Although they speculated that these local differences may have arisen from differences in the local level of expression of TGF-b2 and endogenous osteocalcin expression, the present findings show that these results may reflect the differential expression level of the TGF-b type II receptor and ligand-binding capacity of undifferentiated mesenchyme between the endosteal surface and the trabecular bone. In conclusion, the target cells of TGF-b in normal bone tissue could be considered primarily as extracellular-matrix-producing chondrocytes and undifferentiated preosteoblasts and, as a result, TGF-b may affect matrix production and differentiation of these cells. Although more direct evidence is required to elucidate the roles of TGF-bs in osteoblast and chondrocyte function, the cell surface expression of TGF-b type II receptor should be regarded as essential for regulation of bone and cartilage metabolism.
Acknowledgments: The authors express their thanks to Dr. T. Yamamoto, Department of Pathology, Institute of Nephrology, Niigata University School of Medicine, for DNA sequencing. Thanks are also due to Dr. K. Yamazaki and Dr. N. Sugita, Department of Periodontology, Niigata University School of Dentistry, for their technical help and advice. This study was supported in part by Grants-in-Aid for Scientific Research of the Ministry of Education, Science, Sports and Culture of Japan (Nos. 07407054, 08407056, 08877287), a fund for scientific promotion of Tanaka Industries Co., Ltd., Niigata, Japan; and Immunoresearch Laboratories Co., Ltd., Takasaki, Japan.
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Date Received: September 3, 1997 Date Revised: October 1, 1997 Date Accepted: October 1, 1997
ORIGINAL ARTICLES
Immunoreactive Localization of Transforming Growth Factor-b Type II Receptor-Positive Cells in Rat Tibiae Y. KABASAWA,1,2 S. EJIRI,2 Y. MATSUKI,1 K. HARA,1 and H. OZAWA2 Departments of 1Periodontology and 2Oral Anatomy I, Niigata University, School of Dentistry, Niigata, Japan
TGF-b type II receptor is a transmembrane serine/threonine kinase that, upon ligand binding, recruits and phosphorylates a second transmembrane kinase, the type I receptor in the GS domain, as a requirement for signal transduction.14,29 Phosphorylation allows the receptor to propagate the signal to downstream substrates.14,29,30 In the organogenesis of the mouse embryo, TGF-b type II receptor mRNA expression was detected in the undifferentiated supporting mesenchyme in the developing limb and in the vertebral column.13,21 By immunohistochemical observation during mouse organogenesis, the TGF-b type II receptor localized in Meckel’s chondrocytes, in undifferentiated mesenchymal cells around Meckel’s cartilage, and the cartilage and in the mesenchyme in the base of the skull, which subsequently differentiated into bone.8 In chimeric mice using a TGF-b type II receptor (2/2) embryonic stem cell line, TGF-b type II receptor has been reported to be important for normal development in a variety of organs, due to the several types of congenital abnormalities it reveals.20 We investigated the ligand-binding receptor, the TGF-b type II receptor in postnatal rat tibiae, by immunohistochemistry, RT-PCR, and in situ hybridization, and examined, in particular, the role of TGF-b in normal bone tissue.
To identify the target cells of transforming growth factor-b (TGF-b) in normal bone tissue, we examined TGF-b type II receptor expression using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization in young rat tibiae. In the epiphyseal growth plate, the TGF-b type II receptor cDNA was detected by RT-PCR and, alternatively, the TGF-b type II receptor protein and mRNA expression were observed in the chondrocytes in the lower part of the proliferative cell layer, and in maturative and hypertrophic cell layers by immunohistochemistry and in situ hybridization. Of these, proliferative and maturative chondrocytes, in particular, revealed strong mRNA expression. In the cortical bone area, immunoreactivity for the TGF-b type II receptor was detected in the fibroblastic cells near the osteoblasts on the endosteal surface of cortical bone. In conclusion, our findings suggest that target cells of TGF-b in normal bone tissue could be considered mainly as extracellular matrix-producing chondrocytes and undifferentiated preosteoblasts, and TGF-b may affect matrix production and differentiation of these cells. (Bone 22:93–98; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: TGF-b type II receptor; Immunohistochemistry; In situ hybridization; RT-PCR; Rat tibiae; Growth plate.
Materials and Methods
Introduction
3-week-old male Wistar rats, weighing approximately 56.8 g (n 5 11), were anesthetized with 40 mg/kg body weight sodium pentobarbital, and perfused with 4% paraformaldehyde in a 0.1 mol/L phosphate buffer (pH 7.4) for 15 min. Tibiae were dissected and immersed in the aforementioned fixative at 4°C for 2– 8 h, then decalcified with 10% EDTA (pH 7.3) at 4°C for 1 week. Specimens were dehydrated with an increasing concentration of ethanol and embedded in paraffin. Serial sagittal sections were obtained at a thickness of 6 mm and mounted on poly-Llysine-coated slides. Sections were used for hematoxylin-eosin staining.
Tissue Preparation
TGF-b is one of the most abundant growth factors in bone, and may play a critical role in bone remodeling and cartilage growth and metabolism.2,4 An in vivo TGF-b1 injection to the parietal bone of neonatal rats stimulated intramembranous ossification,17,25 whereas an injection to newborn rat femurs promoted endochondral ossification.11 In experimental defects of mature rat calvariae, grafting of TGF-b-coated b-tricalcium-phosphate pellets promoted new bone formation.19 Joyce et al. suggested that TGF-b was synthesized by osteoblasts and chondrocytes throughout rat bone fracture healing.10 In vitro immunohistochemical study of mouse bone tissue showed that immunoreactivity for TGF-b was present in the sealing zone covered by osteoclasts in the bone resorption lacunae and in the extracellular matrix adjacent to the osteoclasts.7
Immunohistochemical Study Immunohistochemistry was carried out according to Hoshi et al.6 with some modifications. To inhibit endogenous peroxidase, the deparaffinized sections were treated with 100% methanol containing 0.3% hydrogen peroxide for 1 h. The sections were immersed in phosphate-buffered saline (PBS) containing 10% low-fat milk protein for 1 h to block nonspecific binding in the tissue, and then incubated with IgG fractions of anti-TGF-b type II receptor (UBI, Lake Placid, NY) at a concentration of approx-
Address for correspondence and reprints: Kohji Hara, D.D.S., Ph.D., Department of Periodontology, Niigata University, School of Dentistry, Gakkocho 2-5274, Niigata 951, Japan. E-mail: [email protected]. ac.jp © 1998 by Elsevier Science Inc. All rights reserved.
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imately 6 mg/mL overnight. Following washing with PBS, sections were incubated with horseradish peroxidase-conjugated goat, antirabbit IgG (Dako, Glostrup, Denmark) diluted to 1:100 at room temperature for 2 h. Immunoreaction was visualized by incubation in 0.05 mol/L Tris-HCl buffer (pH 7.6) containing 0.05% 39,3-diaminobenzidine (Dojin Co., Kumamoto, Japan), 0.066% sodium azide (Wako Chemicals, Osaka, Japan), and 0.01% hydrogen peroxide. The sections were counterstained with hematoxylin. Negative control specimens were reacted with normal rabbit IgG (Dako) at 6 mg/mL instead of primary antibody. RNA Isolation, RT-PCR Total cellular RNA was extracted from the epiphyseal growth plate of the tibiae of 3-week-old Wistar rats (n 5 20). Epiphyseal growth plates were excised from the tibiae using a dissecting microscope. Total RNA extraction was prepared according to the manufacturer’s protocol (Isogen; Nippon gene, Toyama City, Japan).22 100 mg of tissue was mixed with 1 mL of guanidine isothiocyanate-phenol solution (Isogen), followed by the addition of 200 mL of chloroform, and then centrifuged at 12,000g at 4°C for 15 min. Subsequently, the aqueous phase containing RNA was added with 500 mL of isopropanol, kept at 220°C for 30 min, and centrifuged at 12,000g at 4°C for 10 min. The RNA pellet was diluted in 1 mL of 75% ethanol and centrifuged at 7500g for 5 min at 4°C. The recovered RNA was then resuspended in DEPC-treated H2O (DEPC: 0.1% diethylpyrocarbonate; Aldrich, Milwaukee, WI). First-strand cDNA was synthesized according to the method of Yamazaki et al.,31 using M-MLV reverse transcriptase (Gibco BRL, Gaithersburg, MD) and 50 mg/mL random hexanucleotides (Promega, Madison, WI) from 1 mg of total RNA in the reaction buffer (Gibco) containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, and 3 mmol/L MgCl2, supplemented with 40 U of RNase inhibitor (Toyobo, Osaka, Japan), 10 mmol/L dithiothreitol (Gibco), and dNTPs (dATP, dCTP, dGTP, and dTTP, each at 0.5 mmol/L; Takara shuzo, Shiga, Japan). The reaction mixture was incubated at 42°C for 3 h, then heated at 94°C for 5 min. Nucleotide sequences 301–322 (59-GGAAGTCTGCGTGGCCGTGTGG-39) and 578 –599 (59-CTATGGCAATCCCCAGCGGAGG-39) of the rat TGF-b type II receptor cDNA were used as PCR primers.21,27 A polymerase chain reaction was performed according to Sugita et al.22 A 2 mL volume of reverse transcription (RT) reaction mixture was added to 98 mL of PCR buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, dNTPs (each at 0.1 mmol/L; Takara shuzo), 25 pmol of each primer, and 5 U of recombinant Taq DNA polymerase (Takara shuzo). The reaction mixture was overlaid with mineral oil and subjected to PCR incubation for 38 cycles using an automated DNA thermal cycler (MJ Research, Watertown, MA). The PCR reaction profile was as follows21: 1 min denaturation at 96°C; 1 min annealing at 52°C; and 2 min primer extension at 72°C. The PCR end product was loaded on 3% NuSieve 3:1 agarose gel (FMC Bioproducts, Rockland, ME) in 0.5 3 TBE (45 mmol/L Tris-borate, 1 mmol/L EDTA), stained with 0.5 mg/mL ethidium bromide. The DNA molecular weight marker (fX174/HinfI digest, Nippon gene) was run simultaneously. The gel was photographed with Tri-X pan 400 film for black-and-white prints (Eastman Kodak, Rochester, NY). Probes Used for In Situ Hybridization Total RNA was extracted as previously mentioned from 21 day fetal craniofacial tissue (n 5 2). RT-PCR was performed
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according to the method of Sugita et al.22 0.1 mg of total RNA was added to a reaction mixture containing 50 mmol/L TrisHCl (pH 8.3), 100 mmol/L KCl, 10 mmol/L MgCl2, 4 mmol/L dithiothreitol (Life Sciences, St. Petersburg, FL), 60 U of RNase inhibitor (Toyobo), 0.85 mmol/L dNTPs (Takara shuzo), 50 mg/mL random hexadeoxynucleotide primers (Promega), and 34.8 U of avian myeloblastosis virus reverse transcriptase (Life Sciences) in a final volume of 50 mL. After incubation at 42°C for 3 h, the mixture was heated at 94°C for 5 min, then stored at 4°C. A 2 mL volume of RT reaction mixture was added to 98 mL of PCR buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, dNTPs (each at 0.1 mmol/L; Takara shuzo), 25 pmol of each primer, and 5 U of recombinant Taq DNA polymerase (Takara shuzo). The PCR reaction profile was as follows: 1 min denaturation at 96°C; 1 min annealing at 52°C; and 2 min primer extension at 72°C, for 38 cycles. The PCR end product was loaded on 3% NuSieve 3:1 agarose gel (FMC Bioproducts) in 0.5 3 TBE at 100 V for 1.5 h, stained with 0.5 mg/mL ethidium bromide, and a molecular weight species corresponding to the target 299 bp end product was observed (data not shown). The PCR end product was subjected to electrophoresis on 3% agarose gel and then gel purified using the Qiaex Gel Extraction Kit (Qiagen, Hilden, Germany). For the direct insertion of the RT-PCR product into the plasmid vector pCR™ II, the TA Cloning Kit (Invitrogen, San Diego, CA) was used.12,22 The subcloned fragment was confirmed to be identical to published rat TGF-b type II receptor cDNA by DNA sequence analysis using an automated DNA sequencer (Model 373A, Applied Biosystems, Foster City, CA). In vitro transcription and digoxigenin UTP-labeled sense and antisense probes were prepared as described by the manufacturer (Boehringer Mannheim, Biochemica, Mannheim, Germany). In Situ Hybridization In situ hybridization was performed as described previously.15 Deparaffinized sections were washed in PBS and treated with 1.0 mg/mL proteinase K in 20 mmol/L Tris-HCl (pH 8.0) and 10 mmol/L EDTA at 37°C for 10 min. To remove endogenous alkaline phosphatase, sections were immersed in 0.2 mol/L HCl for 10 min, washed in PBS, and soaked in 0.1 mol/L triethanolamine solution (pH 8.0) containing 0.25% acetic anhydride for 10 min to inhibit nonspecific background binding. The specimens were washed with PBS, dehydrated in graded ethanol, and air dried. The probe was used for hybridization at a final concentration of 1.0 mg/mL in a hybridization mixture (50% formamide, 10 mmol/L Tris-HCl, pH 7.6, 200 mg/mL tRNA, 1 3 Denhardt’s solution, 10% dextran sulfate, 600 mmol/L NaCl, 0.25% SDS, and 1 mmol/L EDTA). The specimens were incubated with the probes at 50°C for 16 h in a 50% formamide humid chamber, then successively washed with 2 3 SSC (standard saline-citrate buffer) containing 50% formamide at 55°C for 30 min, TNE (10 mmol/L Tris-HCl, pH 8.0, 500 mmol/L NaCl, 1 mmol/L EDTA) at 37°C for 10 min, with 20 mg/mL RNase A in TNE at 37°C for 30 min, TNE at 37°C for 10 min, once with 2 3 SSC at 50°C for 20 min, and finally twice with 0.2 3 SSC at 50°C. Specific transcripts were detected with anti-digoxigenin-conjugated alkaline phosphatase antibody (Boehringer Mannheim) by the manufacturer’s protocol. The color was developed with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. The sections were counterstained with methylgreen.
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Figure 1. Histology of 3-week-old rat tibia (hematoxylin-eosin staining). P: proliferative chondrocyte layer; M: maturative chondrocyte layer; H: hypertrophic chondrocyte layer. Original magnification 3225.
Results Epiphyseal Growth Plate Immunohistochemistry. In the epiphyseal growth plate of 3week-old rat tibiae, four layers of chondrocytes were distinguished: resting chondrocytes; proliferative chondrocytes; maturative chondrocytes; and hypertrophic chondrocytes. Beneath the layer of hypertrophic chondrocytes, active endochondral ossification was observed (Figure 1). In the epiphyseal growth plate, immunoreactivity for the TGF-b type II receptor was detected in chondrocytes in the lower part of the proliferative cell layer, and also in maturative and hypertrophic cell layers (Figure 2A). At a higher power, immunoreactivity was seen to correspond to the cell membranes of these chondrocytes (Figure 2B). No specific immunoreactivity was detected on the control sections incubated with normal rabbit IgG (Figure 2A). RT-PCR. Using RT-PCR, TGF-b type II receptor mRNA was present in the epiphyseal growth plate of 3-week-old rat tibiae (Figure 3). By agarose-gel analysis of TGF-b type II receptor cDNA, the predicted band of 299 bp was observed. Negative control was obtained by PCR without the prior RT step, confirming that our RT-PCR yielded products derived from mRNAs. In situ hybridization. In the epiphyseal growth plate of 3week-old rat tibiae, TGF-b type II receptor mRNA expression
Figure 2. (A) Immunopositive cells for TGF-b type II receptor in epiphyseal growth plate. P: proliferative chondrocyte layer; M: maturative chondrocyte layer; H: hypertrophic chondrocyte layer. Left: photomicrograph of section using normal rabbit IgG instead of primary antibody. No specific immunoreactivity was observed. Original magnification 3360. (B) Higher power view of right side of (A), the region of the maturative chondrocyte layer. Immunoreactivity corresponding to the cell membrane of chondrocytes was detected. Arrow heads: TGF-b type II receptor-positive cells. Original magnification 3720.
was detected in chondrocytes in the lower part of the proliferative chondrocyte layer, and in the maturative and hypertrophic chondrocyte layers (Figure 4A). Of these, proliferative and maturative chondrocytes, in particular, revealed strong mRNA
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Figure 3. Agarose-gel analysis of TGF-b type II receptor cDNA. M: marker (fX174/HinfI digest, Nippon gene); S: PCR product.
expression. At a higher power, the transcription signal was observed in cytoplasm (Figure 4B). The result generally coincided with immunolocalization of the TGF-b type II receptor in the growth plate. Trabecular Bone Area and Endosteal Surface Immunohistochemistry. In the area of primary trabecular bone subjacent to the growth plate, no specific immunoreactivity was observed (Figure 5A). In the area of the endosteum, immunoreactivity for the TGF-b type II receptor was detected on fibroblastic cells adjacent to the single layer of osteoblasts on the endosteal bone surface (Figure 5B). Control slides using normal rabbit IgG showed no immunoreactivity (Figure 5A,B). As the endosteal surface, immunolocalization was also observed in fibroblastic cells near the osteoblasts or blood vessels, but not in the osteoblasts of the cortical bone area. In the periosteum, only weak staining for TGF-b type II receptor was detected in the flat cells apart from the bone surface (data not shown). In situ hybridization. TGF-b type II receptor mRNA expression was undetected in both primary trabecular bone and cortical bone. Discussion By immunohistochemistry and in situ hybridization, localization of the TGF-b type II receptor protein and mRNA-expressing cells was evident in the lower part of the proliferative chondrocyte layer, and in the maturative and hypertrophic chondrocyte layers. The proliferative and maturative chondrocytes revealed strong mRNA expression, whereas immunoreactivity for the TGF-b type II receptor was more intense in maturative and hypertrophic chondrocytes. This indicates some possible difference in time kinetics between the cytoplasmic TGF-b type II receptor mRNA expression and immunoreactivity on the cell surface during chondrocyte differentiation. In addition, the functional receptor level may be determined by posttranscriptional regulation, or a receptor recycle mechanism. Immunolocalization of TGF-b1 was observed in the proliferative and upper hypertrophic chondrocytes, and in extracellular matrices around hypertrophic chondrocytes in the growth plate of 1-month-old Long–Evans rats, suggesting that TGF-b induced chondrocyte proliferation and regulated protein synthesis.9 In the growth plate of tibiotarsi of 3-week-old chicks, TGF-b localization preceded a marked increase in type II collagen mRNA
Figure 4. (A) TGF-b type II receptor mRNA expression of epiphyseal growth plate. P: proliferative chondrocyte layer; M: maturative chondrocyte layer; H: hypertrophic chondrocyte layer. Left: photomicrograph of section using sense probe. Original magnification 3360. (B) Higher power view of right side of (A), the region of the maturative chondrocyte layer. Original magnification 3720.
expression in maturative chondrocytes, suggesting a role for TGF-b in synthesis induction of the extracellular matrix.26 In the in vitro model of rat epiphyseal chondrocyte differentiation, TGF-b1 stabilized the phenotype of the prehypertrophic epiphyseal chondrocyte by maintenance of expression of type II collagen and aggrecan core protein, and with coordinate inhibi-
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Figure 5. (A) Localization of the TGF-b type II receptor-positive cells in primary trabecular bone (TB). H: lower part of hypertrophic chondrocyte layer. Left: photomicrograph of section using normal rabbit IgG instead of primary antibody. Original magnification 31000. (B) Localization of the TGF-b type II receptor-positive cells in endosteal bone surface. Arrow heads show fibroblastic cells near the osteoblasts. OB: osteoblasts. Left: photomicrograph of section using normal rabbit IgG instead of primary antibody. Original magnification 31000.
tion of matrix metalloproteinases.1 Taken together with our results, TGF-b produced by chondrocytes and stored at the cartilage matrix would regulate the functional differentiation and matrix production of a portion of the proliferative chondrocytes, and of maturative and hypertrophic chondrocytes, via these TGF-b type II receptors in an autocrine/paracrine fashion during endochondral ossification in the growth plate. In contrast to the growth plate, in the cartilage tissue of the mouse embryo, TGF-b type II receptor mRNA and TGF-b type II receptor-positive cells were detected in the undifferentiated mesenchyme but not in the condensing chondrocytes, suggesting that TGF-b signaling through the type II receptor may play a role in the physiology of these undifferentiated cells.8,13,21 However, based on our findings, the role of TGF-b during the cartilage formation stage may be considered to differ from chondrocyte differentiation in the growth plate. Otherwise, the effect of TGF-b would occur via a different type II receptor isoform, which would be transcribed from the originally identical TGF-b type II receptor gene,5,23 because our primers could not detect these isoforms. Several in vivo and in vitro studies have shown the importance of TGF-b in bone remodeling.2,7,16,18,26,28 Immunoreactivity for TGF-b has been observed in osteoblasts and bone matrix.7,26 Concerning bone matrix protein, TGF-b has been
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found to regulate the synthesis of osteopontin, osteonectin, and collagen in cultured osteoblastic cells,18 whereas it has also been shown to inhibit differentiation-associated phenotypes such as alkaline phosphatase activity and the production of osteocalcin.2,16,18,28 Our immunohistochemical study demonstrates that TGF-b type II receptor was detected in fibroblastic cells adjacent to the osteoblasts on the endosteal bone surface. These immunopositive cells were localized in a well-defined pattern, with the fibroblastic cells particularly close to plump osteoblasts, which appeared to be actively producing bone matrix. On the other hand, no apparent staining was observed in the osteoblasts after terminal differentiation. It was reported that, in cultured MC3T3-E1 cells, TGF-b type II receptor was downregulated in differentiation, suggesting that downregulation is one of the mechanisms by which cells escape from the control of TGF-b.24 Together with our results, these data support a view that, in normal bone tissue, TGF-b would preferentially affect undifferentiated preosteoblastic cells rather than differentiated osteoblasts in regard to extracellular matrix production and differentiation. Most TGF-b response via TGF-b type II receptors may be involved in the regulation of preosteoblast function, and upregulation of the TGF-b type II receptor would be related to rapid bone remodeling. Compared to immunopositive fibroblastic cells for TGF-b type II receptors on the endosteal bone surface, fibroblastic cells adjacent to the osteoblasts did not show similar immunoreactivity in trabecular bone. Consistent with these results, Erlebacher et al. reported that, in the mouse model of osteoblast-specific overexpression of TGF-b2 from the osteocalcin promotor, longitudinal sections through the diaphysis revealed large unmineralized areas of cortical bone in transgenic bone whereas wildtype bone was entirely mineralized. There was, however, no difference in the metaphyseal trabeculation adjacent to the growth plate.3 Although they speculated that these local differences may have arisen from differences in the local level of expression of TGF-b2 and endogenous osteocalcin expression, the present findings show that these results may reflect the differential expression level of the TGF-b type II receptor and ligand-binding capacity of undifferentiated mesenchyme between the endosteal surface and the trabecular bone. In conclusion, the target cells of TGF-b in normal bone tissue could be considered primarily as extracellular-matrix-producing chondrocytes and undifferentiated preosteoblasts and, as a result, TGF-b may affect matrix production and differentiation of these cells. Although more direct evidence is required to elucidate the roles of TGF-bs in osteoblast and chondrocyte function, the cell surface expression of TGF-b type II receptor should be regarded as essential for regulation of bone and cartilage metabolism.
Acknowledgments: The authors express their thanks to Dr. T. Yamamoto, Department of Pathology, Institute of Nephrology, Niigata University School of Medicine, for DNA sequencing. Thanks are also due to Dr. K. Yamazaki and Dr. N. Sugita, Department of Periodontology, Niigata University School of Dentistry, for their technical help and advice. This study was supported in part by Grants-in-Aid for Scientific Research of the Ministry of Education, Science, Sports and Culture of Japan (Nos. 07407054, 08407056, 08877287), a fund for scientific promotion of Tanaka Industries Co., Ltd., Niigata, Japan; and Immunoresearch Laboratories Co., Ltd., Takasaki, Japan.
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Date Received: September 3, 1997 Date Revised: October 1, 1997 Date Accepted: October 1, 1997